Figure S7 from SPP1 Drives Colorectal Cancer Liver Metastasis and Immunotherapy Resistance by Stimulating CXCL12 Production in Cancer-Associated Fibroblasts

Figure S7 Tumor-derived SPP1 promotes the progression of CRLM and immunotherapy resistance independent of macrophages. A Flowchart of the intrasplenic injection model of liver metastasis in mice using OE-SPP1 or OE-vector MC38 cells. i.s.v., intrasplenic injection; i.p., intraperitoneal injection. Clo: Clodronate-liposomes. B Representative H&E staining of metastatic livers from C57BL/6J mice intrasplenically implanted with SPP1-overexpressing or vector-overexpressing MC38 cells, followed by treatment with anti-PD-1 antibody. Scale bar, 1 mm. C Quantification of liver weight and tumor burden in liver metastases from the indicated mouse groups (n = 5 per group), with tumor burden assessed by the tumor-to-liver area ratio in tissue sections. D Flow cytometric analysis of macrophage populations in liver metastases from the indicated mouse groups (n = 5 per group). Mac+ represents the control group, MacΔ represents the macrophage depletion group. E, F Flow cytometric analysis of IFN-γ+ CD8+ and GZMB+ CD8+ T cell populations in liver metastases from the indicated mouse groups (n = 5 per group). G, H Collagen content was evaluated by Masson’s trichrome staining, α-SMA expression levels were assessed via IHC analysis, and cell apoptosis was measured by TUNEL staining. Integrated Optical Density (IOD) was used for quantification in all assays (n = 5 per group). Scale bar = 40 μm. I Flow cytometry validated the engraftment of hCD45+ cell 7 days post implantation in each group of mice (n = 3/group). All results are presented as mean ± SEM. P values were determined by one-way ANOVA. ns, no significant difference, *P < 0.05, **P < 0.01, ***P < 0.001.